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United States Patent |
5,661,020
|
Snow
,   et al.
|
August 26, 1997
|
Low diol polyalkylene oxide biologically active proteinaceous substances
Abstract
Disclosed are pharmaceutical compositions containing low diol polyethylene
glycol, covalently attached to superoxide and dismutase process of making
the compositions. Also disclosed is a method of treatment of disease
processes associated with the adverse effects on tissue of superoxide
anions, such as ischemic events, reperfusion injury, trauma and
inflammation.
Inventors:
|
Snow; Robert A. (West Chester, PA);
Ladd; David L. (Wayne, PA);
Hoyer; Denton W. (Exton, PA)
|
Assignee:
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Sanofi (Paris cedex, FR)
|
Appl. No.:
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632300 |
Filed:
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April 15, 1996 |
Current U.S. Class: |
435/188; 424/94.4; 514/886 |
Intern'l Class: |
C12N 009/96; A61K 038/44 |
Field of Search: |
435/188,181
|
References Cited
U.S. Patent Documents
4179337 | Dec., 1979 | Davis et al. | 435/181.
|
5532150 | Jul., 1996 | Davis et al. | 435/188.
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Foreign Patent Documents |
0 200 467 | Jan., 1979 | EP.
| |
Other References
Abuchowski, A. et al., Cancer Biochem. Biophyscis, vol. 7, pp. 175-186
1984.
Abuchowski, A., Doctor of Philosophy Thesis, "Effects of convalent
Attachment of Polyethylene Glycol on Bovine Serum Albumin and Bovin Liver
Catalase", Rutgers University, New Brunswick, NJ. USA Oct. 1975.
Veronese, F. M., et al., J. of Pharm. & Pharmacol., 35:757-758 1983.
Veronese, F. M., et al. J. of controled Release, 10:145-154 1989.
Zalipsky, S., et al., Eur. Polym. J., 12:1177-1183 1983.
Kazo, G. M., Master of Science in Biochemistry Thesis, "Modification of
Proteins with Activated polyethylene Glycols", Rutgers University, New
Brunswick, NJ Oct. 1985.
|
Primary Examiner: Rollins; John W.
Attorney, Agent or Firm: Davis; William J., Dupont; Paul E., Balogh; Imre
Parent Case Text
This application is division of application Ser. No. 08/245,999, filed May
19, 1994, now U.S. Pat. No. 5,532,150, which is a continuation-in-part of
application Ser. No. 07/936,416, filed on Aug. 27, 1992, now abandoned.
Claims
What is claimed is:
1. A process of preparing a biologically active proteinaceous composition
comprising the steps of:
a) carboxylating polyethylene glycol containing less than 10% w/w
non-monomethoxylated polyethylene glycol;
b) activating said carboxylated polyethylene glycol to obtain an active
polyethylene glycol ester; and
c) covalently attaching said active polyethylene glycol ester to superoxide
dismutase.
2. The process of claim 1 whereto said biologically active protein contains
reactive amino groups thereon.
3. The process of claim 1 wherein the number of polyethylene glycol active
esters attached to said biologically active protein is less than or equal
to the number of reactive amino groups present on said biologically active
protein.
4. A method of treating a disease condition caused by superoxide anions on
tissue in a mammal comprising administering an effective amount of a
composition comprising:
polyethylene glycol having a molecular weight of from about 1,000 to about
15,000 daltons and consisting essentially of less than about 10% w/w
non-monomethoxylated polyethylene glycol and at least about 90% w/w
monomethoxylated polyethylene glycol covalently attached to superoxide
dismutase;
said proteinaceous composition having an immunoreactivity of less than 50%
to mammalian antibodies than the immunoreactivity to mammalian antibodies
of a proteinaceous composition comprising: polyethylene glycol having a
molecular weight of from about 1,000 to about 15,000 daltons and
consisting essentially of more than 10% w/w non-monomethoxylated
polyethylene glycol and less than 90% w/w monomethoxylated polyethylene
glycol covalently attached to superoxide dismutase.
5. The method of claim 4 wherein said disease condition is inflammation.
6. The method of claim 4 wherein said disease condition is ischemia.
7. The method of claim 4 wherein said disease condition is reperfusion
injury.
8. The method of claim 4 wherein said disease condition is trauma.
9. A method of treating a disease condition caused by superoxide anions on
tissue in a mammal comprising administering an effective mount of a
composition comprising:
polyethylene glycol having a molecular weight of from about 1,000 to about
15,000 daltons and consisting essentially of less than about 10% w/w
non-monomethoxylated polyethylene glycol and at least about 90% w/w
monomethoxylated polyethylene glycol covalently attached to superoxide
dismutase;
said composition having an immunoreactivity to mammalian antibodies of from
about 50% to about 1,000% less than the immunoreactivity to mammalian
antibodies of a composition comprising: polyethylene glycol having a
molecular weight of from about 1,000 to about 15,000 daltons and
consisting essentially of more than 10% w/w non-monomethoxylated
polyethylene glycol and less than 90% w/w monomethoxylated polyethylene
glycol covalently attached to superoxide dismutase.
10. The method of claim 4 wherein said disease condition is inflammation.
11. The method of claim 9 wherein said disease condition is ischemia.
12. The method of claim 9 wherein said disease condition is reperfusion
injury.
13. The method of claim 9 wherein said disease condition is trauma.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to improved composition of matter containing
polyalkylene oxide and a biologically active proteinaceous substance,
process of making the composition of matter and method of using the same
for the treatment of disease processes associated with various
physiological disorders in which the administration of the biologically
active proteinaceous substance effects an immune response.
More particularly, this invention relates to improved composition of matter
containing polyethylene glycol-superoxide dismutase, process of making the
composition of matter and method of using the same for the treatment of
disease processes associated with the adverse effects on tissue of
superoxide anions, such as ischemic events, reperfusion injury, trauma and
inflammation.
2. Reported Developments
Biologically active proteins, particularly enzymes and peptide hormones,
have been long considered as ideal drugs for the treatment of various
diseases due to their specificity and rapid catalytic action. Such enzymes
include:
Oxidoreductases such as: Urate: oxygen oxidoreductase (1.7.3.3; "uricase");
Hydrogen-peroxide: hydrogen-peroxide oxidoreductase (1.11.1.6;
"catalase"); Cholesterol, reduced--NADP: oxygen oxidoreductase
(20-.beta.-hydroxylating) (1.14.1.9; "Cholesterol 20-hydroxylase").
Transferases such as: UDP glucuronate glucuronyl-transferase (acceptor
unspecific) (2.4.1.17; "UDP glucuronyltransferase"); UDP glucose:
.alpha.-D-Galactose-1-phosphate uridylyltransferase 2.7.7.12).
Hydrolases such as: Mucopeptide N-acetylmuramyl-hydrolase (3.2.1.17;
lysozyme); Trypsin (3.4.4.4); L-Asparagine aminohydrolase (3.5.1.1;
"Asparaginase").
Lyases such as: Fructose-1,6-diphosphate D-glyceraldehyde-3-phosphate-lyase
(4.1.2.12; "aldolase").
Isomerases such as D-Xylose ketol-isomerase (5.3.1.5; xylose isomerase) and
Ligases such as: L-Citrulline: L-aspartate ligase (AMP) (6.3.4.5).
The peptide hormones include:
Insulin, ACTH, Glucagon, Somatostatin, Somatotropin, Thymosin, Parathyroid
hormone, Pigmentary hormones, Somatomedin, Erythropoietin, Luteinizing
hormone, Chorionic Gonadotropin, Hypothalmic releasing factors,
Antidiuretic hormones, Thyroid stimulating hormone, Calcitonin and
Prolactin.
However, with minor exceptions, enzyme therapy, particularly with non-human
enzymes, has been less than successful due in part to the enzymes'
relatively short half-lives and to their respective immunogenicities. Upon
administration, the host defense system responds to remove the foreign
enzymes by initiating the production of antibodies thereto, thereby
substantially reducing or eliminating their therapeutic efficacies.
Repeated administration of foreign and of otherwise short lived human
enzymes is essentially ineffective, and can be dangerous because of
concomitant allergic response. Various attempts have been taken to solve
these problems, such as through microencapsulation, entrapment in
liposomes, genetic engineering and attachment of the enzymes to polymers.
Among the attempts the most promising appears to be the chemical
attachment of the proteinaceous substances to polyalkylene oxide (PAO)
polymers and particularly polyethylene glycols (PEG). The following
illustrates these attempts.
U.S. Pat. No. 4,179,337 discloses the use of polyethylene glycol or
polypropylene glycol coupled to proteins to provide a physiologically
active non-immunogenic water soluble polypeptide composition in which the
polyethylene glycol (hereinafter sometimes referred to as PEG) serves to
protect the polypeptide from loss of activity without inducing substantial
immunogenic response. The methods described in the patent for the coupling
of polyethylene glycol to a protein involve either the conversion of a
protein amino group into an amide or pseudoamide, with consequent loss of
charge carrying capacity of the amino group, or the introduction at the
amino group of the protein, or vicinal to it, of a heteroatom substituent
such as a hydroxyl group or of a ring system that is not repeated in the
polymer backbone.
Veronese, F. M., Boccu, E., Schaivon, O., Velo, G. P., Conforti, A.,
Franco, L., and Milanino, R., in Journal of Pharmacy and Pharmacology, 35,
757-758 (1983), reported that when bovine erythrocyte derived superoxide
dismutase is modified with a polyethylene glycol carboxylic acid
N-hydroxysuccinimide active ester, the half-life of the enzyme in rats is
increased over that of the unmodified protein.
European Patent Application 0 200 467 of Anjinomoto, Inc. describes
superoxide dismutase that is chemically modified by a polyalkylene oxide
(PAO) which is functionalized at both ends of the polymer with activated
carboxyl coupling groups, each capable of reacting with protein. Because
the activated coupling sites are located at opposite ends of the polymer
chain, it is unlikely that the presence of an activated group at one end
of the polymer can have a significant effect on the reactive nature of the
group at the other end of the polymer. These polymers are capable of
reacting at both ends to cross-couple with proteins to form copolymers
between the protein and the polyalkylene oxide. Such copolymers do not
have well defined or molecularly stoichiometric compositions.
Veronese, F. M. et al in Journal of Controlled Release, 10, 145-154 (1989)
report that the derivatization with monomethoxypolyethylene glycol
(hereinafter sometimes referred to as MPEG) of superoxide dismutase
(hereinafter sometimes referred to as SOD) gives a hererogenous mixture of
products. Heterogeneity was demonstrated to depend on the presence of
bifunctional polyethylene glycol (DPEG) in the monofunctional methoxylated
molecules.
These attempts, in general, have resulted in longer half-life and reduced
immunogenicity of the proteinaceous biologically active substances.
However, further improvements are needed in order to successfully treat a
variety of diseases with these promising biologicals.
We have now discovered that biologically active proteinaceous substances
can be made to possess longer half-life and less immunogenic properties by
chemically modifying them using low diol polyalkylene oxide, particularly
low diol polyethylene glycol (hereinafter sometimes referred to as LDPEG).
The invention will be described with specific reference to SOD, however, it
is to be understood that other biologically active substances may also be
chemically modified using low diol polyalkylene oxides (hereinafter
sometimes referred to as LDPAO).
SUMMARY OF THE INVENTION
In accordance with the present invention, the half-life of biologically
active proteins is increased and their immunogenicity is reduced or
eliminated by covalent modification of the protein with low diol
polyalkylene oxide, preferably low diol polyethylene glycol, employing a
polyethylene glycol active ester intermediate.
Polyethylene glycol exists as a mixture of two forms:
One form contains one --OH group:
##STR1##
This form is called methoxylated, or more specifically, monomethoxylated
polyethylene glycol since it contains one methoxy group per one molecule
of polyethylene glycol.
The other form contains two OH groups.
##STR2##
This form is called the "diol" form of polyethylene glycol since it
contains two --OH groups per one molecule of polyethylene glycol.
The chain-length of the polyethylene glycol depends on the magnitude of n:
the larger the n, the higher the molecular weight.
We have found that polyethylene glycol polymers having average molecular
weights of from about 1,000 to about 15,000 daltons and containing not
more than about 10% w/w of non-monomethoxylated polyethylene glycol are
especially suitable for covalent attachment to biologically active
proteins, especially to superoxide dismutase. More preferably,
polyethylene glycols having average molecular weights of from about 2,000
to about 10,000 daltons and most preferably of from about 4,000 to about
6,000 daltons are used in the present invention wherein the polyethylene
glycol preferably contains less than about 7% w/w and most preferably less
than about 5% w/w non-monomethoxylated polyethylene glycol.
In the process of the present invention, low diol polyethylene glycol is
covalently attached to the biologically active protein as shown
schematically:
a) LDPEG+carboxylating agent.fwdarw.LDPEG-COOH
b) LDPEG-COOH+carboxyl group activating agent.fwdarw.active ester of
LDPEG-COOH
c) n (active esters of LDPEG-COOH)+Protein.fwdarw.(LDPEG-CO).sub.n -Protein
wherein:
LDPEG-COOH is LDPEG carboxylated at hydroxyl sites; and n is the number of
sites of attachment of LDPEG to protein.
LDPEG is carboxylated at the hydroxyl sites, then the carboxyl groups are
esterified with a carboxyl activating agent to form the active esters
which are then coupled to the protein molecule. The number of LDPEG
molecules attached to the protein will vary according to the number of
reactive groups, such as amino groups, present on the protein molecule.
The process of the present invention is applicable to a broad range of
biologically active proteins as well as some peptide hormones. The
biologically active proteins include:
Recombinant human interleukin-4 (rhuIL-4);
Protease Subtilisin Carlsberg;
Superoxide dismutases such as bovine, human, and various recombinant
superoxide dismutases such as recombinant human superoxide dismutase
(rhuSOD);
Oxidoreductases such as: Urate: oxygen oxidoreductase (1.7.3.3; "uricase");
Hydrogen-peroxide: hydrogen-peroxide oxidoreductase (1.11.1.6;
"catalase"); Cholesterol, reduced--NADP: oxygen oxidoreductase
(20-.beta.-hydroxylating) (1.14.1.9; "Cholesterol 20-hydroxylase");
Transferases such as: UDP glucuronate glucuronyl-transferase (acceptor
unspecific) (2.4.1.17; "UDP glucuronyltransferase"); UDP glucose:
.alpha.-D-Galactose-1-phosphate uridylyltransferase 2.7.7.12);
Hydrolases such as: Mucopeptide N-acetylmuramyl-hydrolase (3.2.1.17;
lysozyme); Trypsin (3.4.4.4); L-Asparagine aminohydrolase (3.5.1.1;
"Asparaginase");
Lyases such as: Fructose-1,6-diphosphate D-glyceraldehyde-3-phosphate-lyase
(4.1.2.12; "aldolase");
Isomerases such as D-Xylose ketol-isomerase (5.3.1.5; xylose isomerase) and
Ligases such as: L-Citrulline: L-aspartate ligase (AMP) (6.3.4.5).
The peptide hormones include:
Insulin, ACTH, Glucagon, Somatostatin, Somatotropin, Thymosin, Parathyroid
hormone, Pigmentary hormones, Somatomedin, Erythropoietin, Luteinizing
hormone, Chorionic Gonadotropin, Hypothalmic releasing factors,
Antidiuretic hormones, Thyroid stimulating hormone, Calcitonin and
Prolactin.
A preferred embodiment of the present invention comprises the enzyme
superoxide dismutase covalently attached to low diol PEG. As used herein,
the term "low diol" with respect to a polyalkylene oxide such as
polyethylene glycol refers to a linear polyalkylene oxide containing not
more than about 10% of non-monoalkoxylated polyalkylene oxide, and
preferably not more than about 10% non-monomethoxylated polyethylene
glycol. Stated another way, with respect to polyethylene oxide the term
"low diol" means that more than 90% of the polyethylene glycol is
monomethoxylated.
Superoxide dismutase is an intracellular enzyme present in all
oxygen-metabolizing cells and is responsible for catalyzing the conversion
of the superoxide radical to oxygen and hydrogen peroxide. The superoxide
radical and species derived from it are believed to be causative agents in
a wide variety of inflammatory disorders. Superoxide dismutase is being
used to treat certain inflammatory conditions under the tradename of
Orgotein. In addition, the use of SOD has been investigated for
bronchopulmonary dysplasia and hyperbaric oxygen toxicity, acute
inflammation caused by burns and infections, reperfusion injury following
organ transplants, retrolental fibroplasia, side effects of therapeutic
ionization radiation and certain dermatological conditions. However, when
SOD is administered by intravenous injection to a mammal, the enzyme's
half-life is only a few minutes and it disappears from circulation. As a
result, the enzymatic activity is not sufficient to remove toxic
substances from the bloodstream. Repeated administration on the other hand
causes adverse reactions.
Low diol polyalkylene oxide (LDPAO) comprising chains of polyalkylene oxide
of varying molecular weight and containing at least one hydroxyl group per
chain, such as low diol polyethylene glycol (LDPEG) is attached to
superoxide dismutase (SOD) to form a biologically active composition
having longer half-life and less immunogenicity than either native SOD or
a PAO-SOD composition.
The process of attaching LDPEG to SOD (sometimes hereinafter referred to as
LDPEGation) comprises the steps of:
activating low diol methoxy-PEG, having an average molecular weight of from
about 1,000 to about 15,000, more preferably of from about 2,000 to
10,000, and most preferably from about 4,000 to 6,000 daltons, containing
not more than about 10% non-monomethoxylated PEG, by succinylation to form
LDPEG-succinate (LDPEG-S), preferably with succinic anhydride (SA),
followed by the formation of a reactive ester, preferably with N-hydroxy
succinimide (NHS), to form LDPEG-SS, and then reacting of LDPEG-SS with an
accessible reactive site on SOD, preferably a primary amine residue on
SOD, mainly lysine epsilon amine.
Referring specifically to LDPEG-SOD, the process is as shown:
##STR3##
wherein: LDPEG-OH=low diol CH.sub.3 O-PEG-OH containing not more than
about w/w of HO-PEG-OH
LDPEG-SS=low diol CH.sub.3 O-PEG-OCOCH.sub.2 CH.sub.2 COO(C.sub.4 H.sub.4
NO.sub.2) containing not more than 10% of [(C.sub.4 H.sub.4 O.sub.2
N)OOC-CH.sub.2 CH.sub.2 COO].sub.2 PEG
LDPEG-S=low diol CH.sub.3 O-PEG-OCOCH.sub.2 CH.sub.2 COOH containing not
more than 10% of [HOOC-CH.sub.2 CH.sub.2 -COO].sub.2 PEG
DCC=dicydohexylcarbodiimide
SA=succinic arthydride
bSOD=Bovine Superoxide Dismutase
NHS=(C.sub.4 H.sub.4 NO.sub.2)OH, N-hydroxysuccinimide
(LDPEG).sub.n bSOD=low diol(CH.sub.3 O-PEG-OCOCH.sub.2 CH.sub.2 CO).sub.n
-bSOD
(LDPEG).sub.n-1 bSOD-S=low diol(CH.sub.3 O-PEG-OCOCH.sub.2 CH.sub.2
CO).sub.n -bSOD-COCH.sub.2 CH.sub.2 COOH
SAcid=Succinic Acid
n=number of low diol PEGs per SOD
K.sub.1, K.sub.obs, k.sub.2 and k.sub.3 are rate constants for the
reactions.
LDPEG-SOD is formed by the covalent attachment of activated LDPEG to
reactive sites on SOD, primarily reactive amine sites such as epsilon
lysine statue sites. The latter are converted to an amide for each LDPEG
attached. The resulting LDPEG-SOD product is heterogeneous with respect to
both the degree of pegation and the site of attachment of the LDPEG since
the reaction may occur at least at any number of available reactive amine
sites. In addition, when the LDPEG is bifunctional, i.e., when the
activated reagent is derived from LDPEG did, each end of the LDPEG moiety
can potentially react with reactive sites on the protein. The local
chemical environment proximal to the protein-S-LDPEG linkage site is
essentially independent of the presence of a functional group at the
opposite end of the LDPEG chain so that SOD-S-LDPEG-X would, proximal to
the SOD-S end of the moiety, be independent of X being OCH.sub.3 or OH or
O-Succinate or O-S-SOD. In this regard, antibodies raised to SOD-S-LDPEG-X
should perceive no differences in the regions proximal to protein in
SOD-S-LDPEG versus SOD-S-LDPEG-S-SOD because of symmetry. This would also
be true for the species SOD-S from SOD-S-LDPEG versus from
SOD-S-LDPEG-S-SOD. Indeed, the symmetrical nature of a long chain
polyethylene oxide would require that antibodies that recognized the
ethylene oxide segment would not be able to differentiate between CH.sub.3
O-LDPEG-S-SOD and SOD-S-LDPEG-S-SOD. Only the region of variation at the
terminus of the polyethylene oxide would present a unique epitope for
antibody recognition. Thus it would be expected that antibodies that are
raised to a class of compounds comprised of CH30-LDPEG-S-SOD plus
SOD-S-LDPEG-S-SOD would recognize the CH.sub.3 O-LDPEG end region in a
unique fashion, but would not be able to differentiate between the
LDPEG-S-SOD region uniquely in CH.sub.3 O-LDPEG-S-SOD versus
SOD-S-LDPEG-S-SOD because of the inherent symmetries involved. Thus, in a
system such as the above mixture, were the amount of CH.sub.3
O-LDPEG-S-SOD to be increased relative to the amount of SOD-S-LDPEG-S-SOD
and such a system were then to be exposed to antibodies raised to the
former system, an increased response to the CH.sub.3 O-LDPEG epitope would
be expected in the presence of a constant response to the LDPEG-S-SOD
epitope by the antibodies.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A represents size Exclusion High Performance Liquid Chromatography of
high diol PEG-SOD;
FIG. 1B represents size Exclusion High Performance Liquid Chromatography of
low diol PEG-SOD;
FIG. 1C represents size Exclusion High Performance Liquid Chromatography of
Native SOD;
FIG. 2 represents the effect of diol content in PEG-SS and amount of high
molecular weight (HMW) PEG-SOD produced;
FIG. 3. represents the elution profile of high diol PEG-SOD from Superose 6
Prep. Grade Column using Low Pressure Size Exclusion Chromatography
(LPSEC);
FIGS. 4A-4C represent Size Exclusion High Pressure Liquid Chromatography
(SEHPLC) profiles of the pools obtained in FIG. 3;
FIG. 5 represents the elution profile of low diol PEG-SOD from Superose 6
Prep Grade Column using Low Pressure Size Exclusion Chromatography
(LPSEC);
FIGS. 6A-6C represent Size Exclusion High Pressure Liquid Chromatography
(SEHPLC) profiles of the pools obtained in FIG. 5;
FIG. 7 represents melting temperature values of Pools 1 and 3 (LMW)
fractions of high diol PEG-SOD obtained in FIG. 5;
FIGS. 8A-8H represent Size Exclusion High Pressure Liquid Chromatography
(SEHPLC) of unfractionated high diol PEG-SOD, Pool 1 (HMW), Pool 2 and
Pool 3 (LMW) obtained in FIG. 3 at the start of the study and after 2
weeks exposure at 50.degree. C.;
FIGS. 9A-9I represent profiles of high diol PEG-SOD subjected to base
hydrolysis;
FIGS. 10A-10D represent the elution profiles of PEG products from base
hydrolysis of high diol PEG-SOD (HMW) and low diol PEG-SOD (HMW and (HMW);
FIG. 11 represents sterile/pyrogen-free Superose 6 Prep Grade fractionation
of high diol PEG-SOD; and
FIGS. 12A-12D represent chromatograms of fractions shown in FIG. 11.
DETAILED DESCRIPTION OF THE INVENTION
Different molecular weight components in PEG-SOD prepared from high and low
diol monomethoxy-PEG were isolated and characterized as follows:
Methods routinely used to characterize the molecular weight of proteins,
such as size exclusion chromatography (SEC), ion exchange chromatography
(IEC), and polyacrylamide gel electrophoresis (PAGE) are not well suited
for PEG modified proteins, since the resulting product is a combination of
linear (PEG) and globular (SOD) molecules. In the case of size exclusion
chromatography (SEC), the molecular weight values generated are
`apparent`, since the columns used are designed to fractionate globular
materials, and the presence of an extended linear molecule on the protein
surface makes the entire molecule appear larger than it really is.
Application of IEC to PEG modified proteins is not very useful, since the
extended structure of the PEG groups does not allow intimate interaction
of the protein surface with the solid phase. For native PAGE, the
migrational characteristics are influenced by the charge and the size of
the total molecule, resulting in smeared bands for PEG-SOD after staining.
Sample preparation for SDS-PAGE is quite harsh (SDS treatment in boiling
water), which results in the ester bonds in the linkage between the SOD
and PEG being partially hydrolyzed, resulting in gels which possess no
resolution and extensive smearing. Also, silver staining is not applicable
for pegated proteins, probably due to the interference of the surface
staining of the protein by the PEG groups, and staining has to rely
totally on Coomassie. We have conducted a study to fractionate the
different molecular weight species of PEG modified SOD and characterize
them via Size Exclusion Chromatography (SEC), photon correlation
spectroscopy (PCS), Differential scanning Calorimetry (DSC), high
temperature treatment and base hydrolysis.
Materials and Methods
1. High and Low Diol PEG-SOD
SOD was obtained from DDI, Inc. (Mountainview, Calif.) and pegated using
reagents derived from methoxypoly(ethylene glycol) obtained from Union
Carbide Corporation and comprised of MPEG with high (14-17%) and with low
diol (2.3%) content. Nominal average molecular weights are denoted by
subscripts.
2. Size Exclusion HPLC (SEHPLC)
The solid phase consisted of a Superose 6 Prep Grade HR 10/30 column
(Pharmacia, Inc.). The mobile phase consisted of 50 mM phosphate buffer,
pH 6.2 containing 150 mM sodium chloride. The flow rate was 0.7 ml/min and
the detector was set at 214 nm.
Samples of SOD and PEG-SOD produced with either high or low diol
MeO-PEG.sub.5,000 were injected onto a Superose HR10/30 column (FIG. 1).
SOD (FIG. 1C) possessed a retention time of approximately 24 minutes
corresponding to a molecular weight of 32,000 (calculated from retention
times of molecular weight standards). PEG-SOD made with high diol PEG-SS
(FIG. 1A) produced a bimodal elution profile with the main peak (LMW) at
18.0 minutes (apparent molecular weight: 400,000) and an early eluting
peak (HMW) at 15.4 minutes (apparent molecular weight: 1,000,000), along
with some material eluting in the void volume of the column (exclusion
limit: 40 million MW). The early eluting peak (HMW) for the high diol
PEG-SOD made up to 30% of the total protein based on peak area. PEG-SOD
prepared with low diol PEG-SS (FIG. 1B) produced a bimodal elution profile
with a high molecular weight component comprising less than 6% of the
total protein based on peak area. The retention time for the low diol
PEG-SOD main peak (LMW) was at 18.6 minutes (apparent molecular weight:
370,000) and that of the early eluting peak (HMW) was at 16.7 minutes
(apparent molecular weight: 550,000).
As shown in FIG. 1C, SOD elutes with a retention time of 24.3 minutes which
corresponds to a calculated molecular weight of 34,000. When SOD is
covalently coupled to high diol PEG.sub.5,000, the overall molecular
weight of the resulting material increases. The PEG-SOD prepared with high
diol MeO-PEG.sub.5,000 possesses a bimodal distribution indicating product
heterogeneity (FIG. 1A). The two main peaks elute at 15.4 and 18.0
minutes, comprising 30 and 70% of total protein content based on peak
area, respectively. The early eluting peak (HMW, 15.4 minutes) possesses
an `apparent` calculated molecular weight of approximately 1 million. The
HMW signal also has some material which elutes in the void volume of the
column (10 minutes) corresponding to an `apparent` molecular weight in
excess of 40 million (VHMW). The material from the late eluting peak (LMW,
18.0 minutes) has an `apparent` calculated molecular weight of
approximately 400,000. The true molecular weight of the modified protein
cannot be determined by this type of chromatography, since the column is
measuring `apparent` molecular weight as the solid phase is solely
designed to resolve globular proteins and not linear ones. The presence of
the linear PEG molecule attached to the SOD surface imparts a highly
extended structure to the overall molecule, making the determination of
molecular weight by SEHPLC qualitative at best.
When PEG-SOD is prepared from low diol methoxy-PEG.sub.5,000, the elution
profile is still bimodal (FIG. 1B), but the HMW signal decreases to
approximately 4.8% of the total protein content. Also, the retention times
of the HMW and LMW peaks are now observed to be slightly longer (16.6 and
18.6 minutes for HMW and LMW, respectively) as compared to the high diol
PEG-SOD preparation. This observation, along with the fact that the low
diol preparation does not possess any VHMW material appearing in the void
volume of the column, indicates that lowering the diol content to 2.3%
(from 14-17%) significantly reduces the formation of the high molecular
weight material. It is believed that the VHMW and HMW materials are
primarily formed due to presence of the bis-SS-PEG (formed from the diol
component in the methoxy-PEG) which acts as a crosslinking agent between
proximally oriented lysine groups on separate SOD molecules during
pegation. If this hypothesis is correct, the extent of HMW material formed
should be proportional to the diol content of the MeO-PEG starting
material. In FIG. 2 is shown a plot of the percent HMW signal versus
percent diol for three lots of PEG-SOD, (with 14% diol, 2.3% diol and 1.3%
diol). As seen in the range of diol content studied, there is a nearly
linear increase in percent HMW with diol content supporting the hypothesis
that the extent of HMW material formed in the final product is related to
the diol content of MeO-PEG. Studies indicate that while freshly made
PEG-SOD from the high diol PEG-SS contains approximately 30% HMW material,
after 2 years storage at 5.degree. C., the HMW signal decreases to
approximately 10% indicating that the HMW material is labile and converts
to a lower molecular weight material on storage.
3. Low Pressure Size Exclusion Chromatography (LPSEC)
An XK 50/100 column packed with 1.56 liters of Superose 6 (Prep Grade
Pharmacia, Inc.) was used. The mobile phase consisted of 50 mM phosphate
buffer, pH 6.2, containing 500 mM sodium chloride. The flow rate was 2
ml/min and the detector (Pharmacia, Inc.) was set at 280 nM. Fractions (12
ml) were collected using a Frac-100 Fraction collector (Pharmacia, Inc.)
and analyzed by SEHPLC and pooled.
(a) Fractionation of Components from high diol PEG-SOD
Twenty-five ml of high diol PEG-SOD at 35.3 mg/ml was applied to a 1.59
liter Superose 6 Prep Grade column. The material produced the elution
profile as shown in FIG. 3. SEHPLC analysis of collected samples indicated
that fractions 15 through 30 represent Pool 1 (HMW), fractions 40 through
60 represent Pool 2 and fractions 70 through 90 represent Pool 3 (LMW).
The SEHPLC elution profiles of the pools are shown in FIG. 4.
(b) Fractionation of Components from Low Diol PEG-SOD
Twenty-five ml of low diol PEG-SOD at 28 mg/ml was applied to the 1.59
liter Superose Prep Grade 6 column. The material produced the elution
profile as seen in FIG. 5. SEHPLC analysis of collected samples indicated
that fractions 5 though 15 represent Pool 1 (HMW), fractions 50 through 70
represent Pool 2 (LMW). The elution profiles of the pools on analytical
SEHPLC are shown in FIG. 6.
Fractionating the high diol PEG-SOD preparation on a Superose 6 Prep Grade
column produces an elution profile (FIG. 3), very similar to that observed
with the SEHPLC system used in this study (FIG. 1A). The resulting HMW
pool (FIG. 4B) was not completely free of all LMW material, probably due
to the resolving capacity of the Superose 6 column. Also, LMW from the
high diol PEG-SOD preparation (FIG. 4D) had to be fractionated twice
because the first run still possessed over 15% HMW in the LMW pool. When
the material was re-run, it was possible to decrease the HMW signal to
9.6% of the total protein content. When the fractionated HMW and LMW were
tested for enzyme activity, both were found to be active (data not shown).
In terms of specific activity, there was no detectable inactivation of the
HMW material when compared to unfractionated PEG-SOD or LMW material. It
appears from these studies (and also as reported by Veronese et al,
Journal of Controlled Release, 10:145-154 (1989)) that the observed
decrease in enzyme activity of SOD upon modification with high or low diol
PEG-SS is related to the amino groups which are modified, thereby changing
the charge on the enzyme surface. The size modification induced by the
diol PEG (i.e., formation of HMW) does not lead to any significant
decrease in activity. Whether the HMW material is significantly less
active than other fractions in an absolute sense is still a point for
speculation, since the variability in determining protein concentrations
and enzyme activity introduce considerable error into the data.
When PEG-SOD prepared from low diol PEG-SS is fractionated on the Superose
6 Prep grade column, it produces an elution profile as shown in FIG. 5. As
seen with the high diol PEG-SOD, the low diol PEG-SOD produced an elution
profile which closely resembles chromatograms observed with the analytical
SEHPLC system. The loading conditions for the low diol PEG-SOD were almost
identical to that of the high diol PEG-SOD, but the resulting HMW and LMW
pools (FIGS. 6B and 6C) were almost free of cross-contamination providing
retention times of 15.9 and 18.4 minutes, respectively. It should be noted
that the main peak (LMW) produced in both high and low diol preparations
had very similar retention times (high diol: 18.0 minutes, low did: 18.6
minutes), indicating that the principal product, regardless of diol
content, is similar.
4. Photon Correlation Spectroscopy (PCS)
A Malvern Model 4700 PCS with a 3 watt Spectraphysics laser set at 488 nm
was used for the determination of molecule sizes. The temperatures of the
samples and reference solution were kept at 25.degree. C. SOD (in
deionized water) and PEG-SOD (from high and low diol preparations) were at
concentrations of 10 mg/ml in 50 mM phosphate buffer, pH 6.2, 150 mM
sodium chloride. HMW and LMW samples were at a concentration of 10 mg/ml
in pH 6.2, 50 mM phosphate buffer containing 500 mM sodium chloride.
Samples of (2 ml) were filtered through a 0.2 .mu.m, 25 mm Millipore GV
filter into clean cuvettes and allowed to equilibrate for 15 minutes
before each measurement.
SOD (used as a reference) was observed to have a molecular size of 3 nm
which is in good agreement with literature reported values, while
unfractionated high diol PEG-SOD had a molecular size of 14 nm, HMW & LMW
materials fractionated from high diol PEG-SOD demonstrated molecular sizes
of 22 and 12 rim, respectively. Unfractionated low diol PEG-SOD generated
an average molecular size of 11 nm.
5. Differential Scanning Calorimetry (DSC)
A microcal MC-2 Differential Scanning Calorimeter was used With a starting
temperature of 36.degree. C., a scan rate of 60.degree. C./hr and an
ending temperature of 105.degree. C. SOD and Pool 1 concentrations were at
5 mg/ml while the concentration of Pool 3 (LMW) was at 6.9 mg/ml (both
Pool 1 and 3 were fractionated from high did PEG-SOD). All samples were in
pH 6.2, 50 mM phosphate buffer containing 500 mM sodium chloride. The
samples and reference buffer were filtered through a 0.2 .mu.m filter and
allowed to equilibrate at 35.degree. C. for 30 minutes before the
initiation of scans.
Pool 1 and Pool 3 (LMW) isolated by fractionation of high diol PEG-SOD had
T.sub.m (melting temperature) values of approximately 71.degree. and
91.degree. C., respectively (FIG. 7), while SOD (used as a reference, data
not shown) possessed a melting temperature of 93.degree. C.
6. Sterile/Pyrogen-Free Gel Filtration (S/PF GF)
The same column apparatus as described in Section 3 was used. The column
was pre-washed with 2 liters of 0.1M sodium hydroxide and equilibrated
with at least 10 liters of buffer prefiltered through a 0.2 .mu.m filter
and tested to be free of endotoxins (using a quantitative chromogenic LAL
assay, Whittaker Bioproducts, Inc., P/N 50-647-U). After the column eluent
endotoxin level was found to be less than 1 EU/ml, the elution and pooling
conditions outlined in Section 3 were used.
Twenty-five mls of high diol PEG-SOD applied to a depyrognated 1.56 liter
Superose 6 Prep Grade column produced the elution profile shown in FIG.
11. Pooling of fractions after analytical SEHPLC and rechromatographing
samples provided the chromatograms shown in FIG. 12, (Pool 1 being very
High Molecular Weight (VHMW)), Pool 2 High Molecular Weight (HMW) and Pool
3 Low Molecular Weight (LMW). The recovery (mg of protein) and endotoxin
content of the respective fractions are shown in Table 1.
TABLE 1
______________________________________
Sterile/Pyrogen Free Gel Filtration
Recovery of Different Molecular Weight Components
from High Diol PEG-SOD
Pool Sample Name
MG/ML ML MG EU/ML EU/MG
______________________________________
1 VHMW 1.55 4 6.2 25 16
2 HMW 4.65 14 65 1 0.1
3 LMW 22.2 11.5 255 360 16
______________________________________
7. High Temperature Treatment
Fractionated material from high diol PEG-SOD, as well as unfractionated
high diol PEG-SOD were adjusted to 2 mg/ml in phosphate/salt buffer and
sterile filtered. The samples, in glass vials, were then stressed at
4.degree., 22.degree., 30.degree., 40.degree. and 50.degree. C.
temperature stress conditions. Samples were analyzed by SEHPLC at
predetermined time intervals.
SEHPLC chromatograms of unfractionated high diol PEG-SOD, Pool 1 (HMW),
Pool 2 and Pool 3 (LMW) obtained in FIG. 3 at the start of the study and
after 2 weeks exposure at 50.degree. C. are shown in FIG. 8.
As seen from the thermograms on the HMW and LMW compositions (FIG. 7), the
HMW material has a much lower melting point (71.degree. C. as compared to
91.degree. C.) indicating that the HMW material is significantly more heat
labile. This may explain the conversion of the HMW material to LMW on
storage even under refrigerated conditions. The appearance of secondary
peaks in both thermograms is probably due to the presence of contaminating
LMW in the HMW sample and HMW in the LMW sample (FIGS. 4A and D).
Subjecting the high diol PEG-SOD, HMW, Pool 2 and LMW samples to 50.degree.
C. treatment for two weeks has profound effects on the molecular integrity
of the HMW material as evidenced by the SEHPLC signal (FIG. 8). As can be
seen for high diol PEG-SOD (FIGS. 8A and 8B), the HMW peak decreases from
over 30% of the total protein content to less than 10%, while the
retention time of the main peak increases from 18.3 to 19.1 minutes. For
the fractionated HMW sample, the 50.degree. C. treatment completely
disrupts the original structure of the material and produces an elution
profile which closely resembles PEG-SOD prepared from high diol MeO-PEG
that has been stored for 2 years at 4.degree. C. (data not shown). The
rosin peak for 50.degree. C. treated HMW elutes at 19.3 minutes, and
comprises almost 90% of the total content (FIGS. 8C and D). Similar
effects can be observed on Pool 2 and LMW fractions, where the HMW signal
practically vanishes. Overall, the data from differential scanning
calorimetry and the 50.degree. C. thermal treatment indicate that the HMW
material is heat labile and essentially unstable. These observations can
be extrapolated to storage at 4.degree. C., expecting that the HMW signal
will decrease with time.
8. Base Hydrolysis
HMW and LMW samples as well as unfractionated high diol PEG-SOD, were
subjected to base hydrolysis (pH range 10.8-12.02). The pH of the
solutions was adjusted using 0.1M sodium hydroxide and samples incubated
at room temperature for 2 hours. The samples were then chromatographed
using SEHPLC for protein determination (as outlined in Section 1) using
SOD and succinyl-SOD samples as reference. Free PEG was determined using
the HPLC procedure outlined in Section 6.
Protein Component: The elution profiles of samples of high diol PEG-SOD
subjected to base hydrolysis in the pH range 10.5 to 12.3 (in increments
of 0.3 pH unites) are shown in FIG. 9 (with SOD and Succinyl-SOD as
references).
PEG Component: The elution profile of high diol HMW samples after base
hydrolysis at pH 12.3 is shown in FIG. 10A. The SEHPLC chromatogram of
high diol PEG-SS (5000 MW) is shown in FIG. 10B. Base hydrolysis of low
diol MW and LMW samples produce elution profiles as shown in FIGS. 10C and
10D, respectively. A composite table of retention times of PEG produced
from base hydrolysis of different diol preparations of PEG-SOD, as well as
reference compounds are shown in Table 2.
TABLE 2
______________________________________
PEG CHROMATOGRAPHY
PEG-SOD HMW Base Hydrolysis Products from High and Low Diol
Preparations and Different Molecular Weight PEGs and Retention Times
Sample Diol (H/L)
Retention Time
Peak # %
______________________________________
PEG-SOD, HMW
High 13.19 1 24
High 13.72 2 76
PEG-SOD, HMW
Low 13.19 1 5
Low 13.72 2 95
MeO-PEG 5000
High 13.05 1 28
High 13.59 2 72
MeO-PEG5000
Low 13.19 1 1
Low 13.72 2 99
Peg 8000 * 13.14 1 100
Peg 10000 * 12.82 1 100
______________________________________
*Used as molecular weight marker, being 100% diol.
When high diol PEG-SOD is subjected to different degrees of base hydrolysis
(FIG. 9), a peak that closely corresponds to succinyl-SOD (retention time:
23.8 minutes), begins to appear when the hydrolysis pH is adjusted to
values >10.8 (FIG. 9C). As the pH increases from 10.8 to 12.3, there is a
progressive loss of the original PEG-SOD peak, while a peak that resembles
succinyl-SOD begins to appear. Finally, between pH 12.0 and 12.3 (FIGS. 9F
and G), the hydrolysis seems to be complete and the end product has a
retention time similar to that of succinyl-SOD. Even under mildly basic
conditions (FIG. 9B), the HMW signal is extremely sensitive to base,
indicating that a primary linkage comprising this species is probably an
ester bond. These data are fundamental in characterizing the HMW material.
Though not presented here, unfractionated PEG-SOD and HMW material exposed
to sodium dodecyl sulfate (SDS treatment), remained intact indicating that
HMW material is not held together by ionic bonds. Also, on reduction with
mercaptoethanol in the presence of SDS the HMW material remained intact
indicating that it is not held together by disulfide bonds.
When base hydrolysis is performed on the HMW material from both high and
low dial PEG-SOD, the resulting hydrolysate from each preparation has
significantly different elution profiles for free MeO-PEG (FIG. 10). As
seen in FIG. 10A, the HMW from high dial PEG-SOD produces a PEG peak which
is bimodal in nature, with peaks at 13.2 and 13.7 minutes, corresponding
to molecular weights of 8000 and 5000, respectively (Table 2). The MeO-PEG
used in the pegation reaction has an `average` molecular weight of 5000.
The presence of the 8000 MW PEG is related to dial material which arises
during ethylene oxide polymerization and which leads to formation of a PEG
polymer of higher molecular weight via chain growth at both ends of the
polymer (i.e., to 8000 as compared to 5000). The ratio of the PEG 8000 to
5000 peak areas in hydrolyzed HMW was observed to be approximately 24:76
while the corresponding value for the PEG-SS starting material was 14:86.
Therefore, it is apparent that there is a higher proportion of 8000 MW PEG
in the HMW material. Conversely, when either HMW or LMW fractions from low
dial PEG-SOD are analyzed after base hydrolysis, the prominent peak (i.e.
>95%) corresponds to that of PEG 5000 MW with minor amounts of 8000 MW PEG
(HMW-5% and LMW<1%; FIGS. 10C and D). The 8000 MW PEG contaminant is
already present in the low dial PEG-SS (Table 2), and similar to the
observations made with the high dial PEG-SOD, the 8000 MW PEG seems to
accumulate in the HMW material. It is evident from these studies that the
8000 MW PEG SEHPLC signal can be used as a marker to determine (after base
hydrolysis) the extent of crosslinked PEG-SOD in the preparation.
The study results indicate that the extent of HMW material formed in the
final PEG-SOD product is related to the diol content of the MeO-PEG used
in the synthesis. The SOD concentration used in the reaction mixture may
also influence the extent of HMW material formed (by mass action) but the
underlying mechanism for the formation of HMW appears to be due to
crosslinking of SOD molecules mediated by bis-SS-PEG.
Starting Materials. Intermediates and Reagents
Superoxide Dismutase
Superoxide dismutase is the name given to a class of enzymes that catalyze
the breakdown of the superoxide anion radical (O.sub.2.sup.-.) to oxygen
and hydrogen peroxide.
SOD is known under the systematic nomenclature of the International Union
of Biochemistry as superoxide oxidoreductase and has a classification
number of 1.15.1.1. Such substances have been called orgoteins and
hemocupreins as well as superoxide dismutases and range in molecular
weight from about 4,000 to about 48,000. The copper-zinc dismutases are a
remarkably conserved family with respect to gross structural properties.
Without exception, the purified enzymes have been shown to be dimers
(molecular weight usually 31,000-33,000) containing two moles each of
copper and zinc ions per mole. The enzymes of the manganese/iron family
are not as uniform in such basic properties as molecular weight, subunit
structure and metal content. Some are dimers; others are tetramers. The
content of metal ranges from about 0.5 to 1 mole per mole of subunit
polypeptide chain. Naturally occurring Zn/Cu-containing enzymes from
mammals and their functionally competent analogs and muteins are
considered to be mammalian Zn/Cu superoxide dismutases (mSOD).
In formulations of the present invention mSOD may be of any origin. It is
commercially obtained from bovine erythrocytes and human erythrocytes as
well as by recombinant synthesis in microorganisms, such as E. coli and
yeast. Among other sources, Cupri-Zinc bovine liver superoxide dismutase
(SOD, EC 1.15.1.1) for example, is available from DDI Pharmaceuticals,
Inc. (Mountain View, Calif.).
Polyethylene Glycol
In practicing the present invention, low diol PEG is utilized for
attachment to biologically active proteins. While certain molecular weight
methoxypolyethylene glycols are available commercially (for example,
methoxy-PEG.sub.5,000 was obtained from Union Carbide Corporation in two
forms: a conventionally available high diol methoxy-PEG.sub.5,000 which
contained 14-17% of higher molecular weight PEG diol, and a low diol
product which contained less than 4% PEG diol) some are required to be
made and purified in order to produce a pegated protein that possesses low
immunogenicity. For example, pegation of SOD with methoxy-PEG-SS derived
from some commercial sources leads to a product containing high molecular
weight components, as verified by size exclusion chromatography, discussed
earlier. This high molecular weight product is believed to derive from
protein crosslinking through an activated diester formed from the various
mounts of PEG diol found in the commercial sources of M-PEG. The
individual active esters, although located on the same polymer chain, are
nonetheless chemically remote from one another. Thus, the presence of a
second reactive functionality in the polymer tends to exert an
increasingly negligible effect on the reactivity of a first reactive
functionality as the distance separating the two functionalities
increases. The individual reactivities thus tend to be independent of
moieties present at opposite ends of the polymer chain, and crosslinking
cannot be avoided in the absence of infinite dilution of reagents. It is,
accordingly, important to synthesize an M-PEG-SS known to contain very
small amounts, preferably no amounts of diester. S. Zalipsky et al in the
Journal of Bioactive and Compatible Polymers, Vol. 5, April 1990, pp.
227-231, described the purification of polyethylene glycol 2000 from
methoxypolyethylene glycol 2000. The succinate esters are also prepared
and shown to separate by ion exchange chromatography on DEAE-Sephadex. The
preparative method is shown in Example 4.
While the procedure described in Example 4 works well with PEG-2000, it
fails with higher molecular weight PEG's. Higher molecular weight PEG
acids do not bind to anion or cation resins; the greater mass of
polyethylene backbone is believed to mask any ionic properties of the
pendant acid. We have found that extremely low ionic strength buffer was
required to bind the PEG succinates and they eluted under very low
increases of ionic strength indicating that they are only very weakly held
by the resin.
We have found that higher molecular weight methoxy-PEGs can be separated
from diol components if the hydroxyl functionalities are first converted
to dimethoxytrityl (DMT) ethers before application of reverse phase thin
layer chromatography. The hydroxyls can be liberated by acid treatment.
The schematics of preparation and purification of methoxy-PEG.sub.5000
-dimethoxytrityl (M-PEG-DMT) derivatives are as follows; while the details
are shown in Examples 5 through 8.
M-PEG-DMT and DMT-PEG-DMT are prepared in an identical fashion. The
polyether is dissolved in ethanol-free chloroform and the solution dried
by distilling off approximately half the chloroform at atmospheric
pressure under a blanket of argon. The solution is then allowed to cool to
room temperature under argon, followed by the sequential addition of
excess diisopropylethyl amine (1.5 eq.), 10 mol % 4-dimethylaminopyridine
as catalyst, and finally an excess amount of 4,4-dimethoxytrityl chloride
(1.2 eq.). After 15 hours reaction, the solution is concentrated by rotary
evaporation and the solution added to anhydrous ether to precipitate the
tritylated PEG. Regular phase TLC cleanly separates starting material from
product, the PEG backbone staining with Dragendorfs reagent. While
M-PEG-DMT is not resolved from DMT-PEG-DMT by regular phase TLC, reverse
phase C-18 TLC plates cleanly separate M-PEG-DMT, DMT-PEG-DMT, DMT
chloride and DMT alcohol from each other (mobile phase 4:1:1
acetonitrile/water/isopropanol). PEG backbone is confirmed by staining
orange to Dragendorfs and trityl incorporation confirmed by exposing the
plate to HCl vapors to give an orange stain.
Authentic M-PEG-DMT 5000 was shown to separate cleanly from authentic
DMT-PEG-DMT 8000 on a Waters C-8, 300 angstrom pore size, 15-20 micron
particle size Prep-Pak Bondapak cartridge. The crude M-PEG-DMT was
dissolved by sonication in 30% acetonitrile/water to a concentration of
approximately 12 mg/ml and passed through a 2.5 micron filter. The sample
was loaded onto the column (2 g in 25 ml) in a 30% acetonitrile/water
mobile phase. After 8 minutes of isocratic elution, a contaminating peak
eluted (identity unknown, having a high absorbance at 280 nm but
accounting for very low relative mass). A gradient of 30-70%
acetonitrile/water over 21 minutes was then begun, and the desired
M-PEG-DMT eluted at 58-60% acetonitrile. Authentic DMT-PEG-DMT typically
elutes at 80% acetonitrile. The first 3/4 of the desired peak is collected
and the last 1/4 discarded. In this way, 15.4 g of M-PEG-DMT was purified
from 22.6 g of crude M-PEG-DMT.
The trityl cleavage of M-PEG-DMT is as follows:
Attempted removal of the DMT group from M-PEG-DMT with HCl gave by TLC
(crude undiluted reaction mixture) complete removal of the trityl group.
However, concentration of the chloroform extract gave a back reaction
which resulted in a re-tritylation of a significant portion of the PEG. It
was not possible to purify this by selective precipitation. The hydrated
trityl cation and chloride are apparently in equilibrium with the result
that dehydration, such as occurs during solvent removal, produces
significant quantities of DMT chloride. This re-tritylation may be
prevented by the use of a non-equilibrating counterion. Sulfuric acid was
shown to irreversibly de-tritylate M-PEG-DMT. The sulfuric acid cleaved
M-PEG is extracted into chloroform, concentrated and precipitated into
ether to give pure zero diol M-PEG. In this manner, 10 g of M-PEG-DMT was
cleaved to 8.68 g of zero diol M-PEG. Size exclusion chromatography
indicates this material contains less than 0.3% diol.
Other higher molecular weight methoxy-PEG derivatives can be made by
analogous processes.
The following examples will serve better to illustrate the practice of the
present invention.
EXAMPLE 1
A. Methoxypolyethylene Glycol Succinate (M-PEG-S)
In a 2 liter flask, 100 g (0.02 mole) of methoxy-PEG.sub.5,000 (M-PEG) was
dissolved with stirring in 300 ml of warm (40.degree. C.) anhydrous
toluene. The volume was reduced by azeotropic removal of 147 ml of toluene
under a nitrogen atmosphere to reduce the water content of the m-PEG from
1.73 to 0.23%. After cooling to ambient temperature, 233 ml of dry
methylene chloride followed by 3.0 g (0.09 moles) of succinic anhydride
and 1.1 g (0.01 mole) of 4-diimethylaminopyridine (DMAP) were added. The
reaction was stirred and heated at reflux overnight, and then 200 ml of
methylene chloride was removed at reduced pressure. The residue was added
with stirring to 1.6 liters of ether in a 4 liter flask. This was stirred
for 45 minutes and filtered. The filter cake was washed with 70 ml of
ether and dried at reduced pressure to afford 100.4 g of crude
m-PEG-succinate (m-PEG-S) as a white solid containing DMAP.
The crude M-PEG-S (100 g) was dissolved in 633 ml of methylene chloride and
passed through a column containing 114 g of Dowex 50.times.8-100H+ resin
previously washed with 272 ml dioxane followed by 316 ml of dry methylene
chloride. The column was then washed with an additional 316 ml of
methylene chloride, and the eluents were combined and dried over anhydrous
magnesium sulfate. Methylene chloride (800 ml) was removed under reduced
pressure. The remaining solution was added with stirring to 1600 ml of
ether in a 4000 ml flask. After stirring for 30 minutes, the suspension
was allowed to stand for 30 minutes and then filtered. The filter cake was
then washed with 75 ml of ether and dried at reduced pressure. This
afforded 96.0 g :(94% yield) of m-PEG-S as a white solid which exhibited a
proton NMR spectrum consistent with the assigned structure: .sup.1 H-NMR
(CDCl.sub.3): 4.27 (triplet, 2H, --CH.sub.2 --O--C(.dbd.O)--), 3.68 (large
singlet offscale, PEG methylene O--CH.sub.2 --'s), 3.39 (singlet, 3H,
OCH.sub.3), and 2.65 ppm (narrow multiplet, 4 H, --C(.dbd.O)--CH.sub.2
--CH.sub.2 --C(.dbd.O)--). The carboxylic acid content of 0.000207 mol/g
was measured by titration.
B. Methoxypolyethylene Glycol N-Succinimidyl Succinate (M-PEG-SS)
In a 2,000 ml flask, 98.48 g (0.0192 mole) of methoxypolyethylene glycol
succinate (m-PEG-S) was dissolved in 468 ml of dry toluene with warming to
40.degree. C. The solution was filtered and the volume was reduced by 263
ml by azeotropic distillation under nitrogen. The resultant viscous liquid
was transferred to a 1,000 ml three-necked flask under nitrogen using 225
ml of dry methylene chloride. To this was added 2.22 g (0.0192 mole) of
N-hydroxysuccinimide, and the reaction was stirred until the
N-hydroxysuccinimide dissolved. The reaction mixture was then cooled to
5.degree. C. in an ice bath, and a solution of 4.44 g (0.0125 mole) of
dicyclohexylcarbodiimide (DCC) in 24 ml of methylene chloride was added
dropwise over 5 minutes. During the addition of the methylene chloride/DCC
solution, dicyclohexylurea (DCU) began to crystallize from the reaction
mixture. The reaction was allowed to warm to room temperature and was
stirred overnight. The content of the reaction flask was transferred to a
2,000 ml flask using 25 ml of methylene chloride to rinse the flask. At
reduced pressure at 30.degree. C., 250 ml of methylene chloride was
removed, the suspension was filtered and the filter cake was washed with
25 ml of dry toluene. The filtrate was then added to 1,200 ml of anhydrous
ether with stirring, and the resultant suspension was stirred for 45
minutes before being filtered. The filter cake was rinsed with 100 ml of
dry ether and dried under a latex rubber dam for 2 hours. The resultant
solid was then dried under high vacuum and transferred to a bottle in a
glove bag under argon. This afforded 96.13 g (96.1% yield) of the title
compound (m-PEG-SS) as a white solid which exhibited a proton NMR spectrum
consistent with the assigned structure: .sup.1 H-NMR (CDCl.sub.3): 4.32
(triplet, 2H, --CH.sub.2 --O--C(.dbd.O)--), 3.68 (large singlet offscale,
PEG methylene O--CH.sub.2 --'s), 3.39 (singlet, 3H, OCH.sub.3), 2.99 and
2.80 (pair of triplets, each 2H, succinate --C(.dbd.O)--CH.sub.2 CH.sub.2
--C(.dbd.O)--), and 2.85 ppm (singlet, 4H, succinimide
--C(.dbd.O)--CH.sub.2 CH.sub.2 --C(.dbd.O)--). The active ester content of
the product was determined by reaction with excess benzylamine in toluene
followed by back titration with perchloric acid in dioxane to a methyl red
end-point and found to be 0.000182 mole/g.
C. Low Diol PEG-SOD
11.8 g of an aqueous solution of SOD containing 82.1 mg of protein per gram
was diluted to a total weight of 200 g with 0.1M sodium phosphate buffer
at pH 7.8. To this solution, magnetically stirred and heated to 30.degree.
C., was added 3.4 g of low diol methoxy PEG-SS prepared in Example 1B. The
pH of the reaction mixture was maintained at 7.8 using a Mettler DL25
titrator programmed in the pH stat mode to add 0.5 normal sodium hydroxide
solution as required. After 1 hour the reaction mixture was filtered
through a 0.2 micron low protein binding polysulfone filter, concentrated
to about 60 ml using a stainless steel Millipore Mini-tan device equipped
with a 30,000 NMWL membrane 4 pk and was then subjected to dialfiltration
against 2 liters of 50 mM sodium phosphate buffered saline (0.85%) at pH
6.2 to 6.3. The retentate solution containing the low diol PEG-SOD was
then filtered through a 0.2 micron filter.
EXAMPLE 2
High Diol PEG-SOD
A high diol PEG-SOD was prepared in the same manner as low diol PEG-SOD
using high diol PEG-SS.
EXAMPLE 3
A. Monomethoxypolyethylene glycol succinate
A 12 liter three-neck flask was charged with 4 liters of toluene and 2212 g
of methoxypolyethylene glycol, previously warmed to 70.degree. C. under
nitrogen. The volume was reduced by azeotropically removing 1.3 liters of
toluene at reduced pressure. After cooling to 30.degree. C., there was
added 4 liters of methylene chloride followed by 66.4 g of succinic
anhydride and 24.4 g of 4-dimethylamlnopyridine. The reaction was refluxed
for 32 hours then 3.8 liters of methylene chloride was removed at
atmospheric pressure. The reaction was cooled and poured into a 5 gal.
glass carboy containing 28 liters of methyl tert-butyl ether with
stirring. The resulting suspension was stirred for 1 hour and collected on
a Lapp filter. The filter cake was washed with 1 liter of methyl
tert-butyl ether. Drying in a vacuum over overnight at room temperature
yielded 2.252 kg of the title compound as a crude white solid.
The crude title compound was dissolved in 8 liters of methylene chloride
and passed through a glass pressure column containing 3.0 kg of Dowex
50W-X8 resin (cation exchange, hydrogen form) previously washed with 5
liters acetone followed by 4 liters of methylene chloride. The column was
then washed with 3 liters of methylene chloride. The column eluents were
combined and 10 liters of methylene chloride was removed at atmospheric
pressure. The remaining solution was poured into 26 liters of methyl
tert-butyl ether with stirring. The resulting suspension was stirred for
45 minutes 3rid the solid was removed by filtration. This was washed with
3 liters of methyl tert-butyl ether. Drying in a vacuum oven at room
temperature yielded 2.46 kg of a white solid of the title compound, 95%
recovery. This material contained 1.5% methoxypolyethylene glycol, and
assayed at 2.72.times.10.sup.-4 mole/g (theory is 1.96.times.10.sup.-4
mole/g).
B. Methoxypolyethylene glycol N-succinimidyl succinate
In a 12 liter flask under nitrogen 1.5 kg of monomethoxypoly ethylene
glycol succinate was dissolved in 7.2 liters of toluene with warming. The
volume was reduced by 2.8 liters at reduced pressure to remove water. The
resultant viscous liquid was cooled to 40.degree.-45.degree. C. and 3.4
liters of methylene chloride was added followed by 33.89 g of
N-hydroxysuccinimide. The reaction was stirred for 1 hour until all the
N-hydroxysuccinimide was dissolved, then the reaction was cooled to
10.degree. C. and a methylene chloride solution (368 ml) of 67.75 g
1,3-dicyclohexylcarbodiimide (DCC) was added dropwise over 30 minutes. The
reaction was allowed to warm slowly to room temperature while being
stirred over 18 hours. The volume was then reduced by 3.2 liters at
atmospheric pressure. The suspension was cooled to 0.degree.-5.degree. C.
and stirred for 30 minutes. This was filtered and the filter cake was
washed with 250 ml of toluene. The filtrate and the wash was added to 28
liters of methyl tert-butyl ether with stirring. The resultant suspension
was stirred for 45 minutes and then filtered on a Lapp filter. The filter
cake was washed with 1 liter of methyl tert-butyl ether and dried under a
latex dam for 4 hours. Additional drying at room temperature in a vacuum
oven at reduced pressure overnight yielded 1.5 kg of a white solid, 100%
yield. This material assayed at 1.79.times.10.sup.-4 mole/g (theory is
1.92.times.10.sup.-4 mole/g).
C. Methoxypolyethylene glycol succinoylbovine superoxide dismutase
To 32 liters of warm (29.degree.-30.degree. C.) pH 7.8 phosphate buffer in
a 42 liters reactor containing a pH electrode was added 194.0 g of bovine
erythrocyte superoxide dismutase. The volume was adjusted to 39.5 liters
and the reaction was warmed to 29.degree. C. The sodium hydroxide tube
from the pH titrator was adjusted over the center of the reactor directly
above the surface of the solution. The pH titrator was initiated and the
pH was adjusted to 7.8 with 0.5N sodium hydroxide. At this time 614.7 g of
methoxypolyethylene glycol N-succinimidyl succinate was added over two
minutes and the reaction was stirred for 41 minutes while the pH was being
adjusted to 7.8 with 0.5N sodium hydroxide with the reaction temperature
being maintained at 30.degree. C. The reaction was then filtered through a
200 Millipak filter and concentrated using a Millipore stainless steel
Pellicon diafiltration system. The reactor was then rinsed with 600 ml of
pH 6.2 phosphate buffer. The rinse was added to the concentrate after
filtering through the Millipore 200 filter and the dialfiltration system.
The final volume of the concentrate was about 9 liters. The concentrate
was then diafiltered, using the Millipore Pellicon diafiltration system
against 200 liters of pH 6.2 phosphate buffer over 2.17 hours. The
diafiltration system was rinsed with 1.5 liters of pH 6.2 phosphate
buffer. The final volume of the concentrate was about 8 liters. The
concentrate was then transferred to a clean 5 gal glass carboy through an
inline Millipore 200 Millipak filter and the filter was rinsed with 500 ml
of pH 6.2 phosphate buffer. This afforded 11.98 kg (91.4% yield) of the
title compound as a clear greenish-blue solution. (Activity: 32,960
units/ml).
EXAMPLE 4
A. Preparation of partially carboxymethylated polyethylene oxide
Polyethylene oxide, M.sub.w 2000 (Fluka, 25 g, 25 meq. OH) was dissolved in
toluene (120 ml) and azeotropically dried until no more water appeared in
the Dean-Stark trap attachment (approx. 25 ml of toluene were removed).
The solution was cooled to 50.degree. C. and treated with potassium
tert-butoxide (1.7 g, 15 mmol). The solution was brought to reflux and
more solvent was distilled off (approx. 25 ml). The stirred reaction
mixture was brought to 25.degree. C., and treated overnight with ethyl
bromoacetate (3.4 ml, 16 mmol). The precipitated salts were removed by
gravity filtration, and washed with methylene chloride (30 ml). The
polymer was recovered by partially concentrating the filtrate (to approx.
60 ml), and slowly pouring the concentrated solution into ethyl ether (300
ml) at 5.degree. C. with vigorous stirring. The collected white polymeric
powder was dried in vacuo. Yield: 24g; IR (neat) showed the characteristic
ester absorption at 1753 cm.sup.-1. The polymer was dissolved in 1N NaOH
(50 ml), and NaCl (10 g) was added. After approx. 45 min this solution was
acidified with 6N HCl to pH 3.0 and extracted with methylene chloride
(3.times.60 ml). The combined organic phases were dried (MgSO.sub.4),
concentrated (to approx. 50 ml), and poured into cold stirring ether (300
ml). The precipitated product was collected by filtration and dried in
vacuo. Yield: 22 g; IR (neat) showed absorption at 1730 cm.sup.-1,
corresponding to .omega.-carboxyl group.
B. Preparation of pure .alpha.-hydroxy-.omega.-carboxymethylpolyethylene
oxide by separation of partially carboxymethylated PEO on DEAE-Sephadex
The mixture of homo- and heterobifunctional PEO's (22 g) was dissolved in
water (40 ml), and applied to a column containing DEAE-Sephadex A-25
(Sigma, 27 g, 0.1 mole ion-exchange sites) in the tetraborate form. The
first fraction containing underivatized polymer was eluted with deionized
water. When the eluent became negative to a PAA test, a stepwise ionic
gradient of ammonium bicarbonate (from 6 to 22 mM at increments of 1-2 mM
every 100 ml) was applied, and fraction collection (approx. 40 ml each)
began. Fractions 2-21 were positive to the PAA test, and contained pure
monocarboxylated PEO (R.sub.1 =0.49). The subsequent three fractions did
not contain PEO, while fractions 25-36 contained the pure PEO-diacid
(R.sub.1 =0.26). The fractions containing
.alpha.-hydroxy-.omega.-carboxymethylpolyethylene oxide were combined and
concentrated (to approx. 100 ml). Sodium chloride (35 g) was dissolved in
this solution, which was then acidified to pH 3 and extracted with
methylene chloride (3.times.100 ml). The combined CH.sub.2 Cl.sub.2
solution was dried (MgSO.sub.4), concentrated (to approx. 100 nil), and
slowly poured into cold stirring ether (500 ml). The precipitated polymer
was collected and thoroughly dried in vacuo to give 8.8 g of product.
.sup.13 C-NMR (CDCl.sub.3): .delta. 172.7 (COOH); 72.4 (CH.sub.2 CH.sub.2
OH); 70.4 (PEO); 69.0 (CH.sub.2 COOH); 61.3 (CH.sub.2 OH)ppm.
Bis-carboxymethylpolyethylene oxide isolated from the column was also
analyzed. .sup.13 C-NMR (CDCl.sub.3): .delta. 172.4 (COOH); 70.4 (PEO);
68.8 (CH.sub.2 COOH) ppm.
EXAMPLE 5
Synthesis of dimethoxytrityl methoxypolyethylene glycol
Methoxypolyethylene glycol (5,000 dalton average molecular weight; 36.3 g,
7.26 mmol) was dissolved in 500 ml chloroform, followed by the removal by
distillation of 250 ml chloroform to remove water. A drying tube was
attached to the flask and the solution allowed to cool to approximately
50.degree. C. N,N-diisopropylethylamine (1.8 ml, 10.3 mmol) was added,
followed by 4-dimethylamino pyridine (100 mg, 0.8 mmol, 10 mol %) and 2.9
g of 4,4-dimethoxytrityl chloride (98%).
The mix was allowed to stir overnight at room temperature at which time the
solvent was removed by rotary evaporation at 60.degree. C. The residue was
taken up in a small amount of chloroform, and the M-PEG-DMT was
precipitated by addition into 2 liters of anhydrous ether. The precipatate
was collected, dried and chromatographed on a C-8 300A reverse phase prep
column on a Waters LC4000 system employing a 30-95% acetonitrile gradient
(against water) over 20 minutes. The desired product eluted at 58-60%
acetonitrile. The sample (2 g) in 20 ml of 30% acetonitrile/water was
loaded onto the column at 50 ml/min flow rate. This eluent (30%
acetonitrile/water) was allowed to continue isocratically until a large
impurity peak was eluted, typically 3-5 min, mv 280 .mu.m. After the
elution of this first peak, the gradient was started. The next peak to
elute was the desired methoxy-PEG-DMT 5000. The first 3/4 of the peak was
collected, and the tail end of the peak was discarded.
In this fashion, 22.6 g of crude M-PEG-DMT 5000 was purified in 2 g
portions to obtain 15.44 g of the title product.
EXAMPLE 6
Synthesis of zero diol methoxypolyethylene glycol from dimethoxytrityl
methoxypolyethylene glycol
10 g M-PEG-DMT 5000 was placed in a 500 ml flask and dissolved in 320 ml
Milli-Q water. Sulfuric acid was added (80 ml) as a slow stream to bring
the concentration to 20%. The solution turned red and homogeneous. After
stirring overnight, the acid solution was extracted with 2.times.500 ml
chloroform, and the combined extracts dried over MgSO.sub.4, concentrated,
and the red oil poured as a thin stream into 2 liters of anhydrous ether
at 20.degree. C. The precipitate was allowed to settle for 24 hours. It
was collected in a course frit sintered glass funnel, and then washed with
2.times.200 ml portions of anhydrous ether. The precipitate cake was
broken up and dried under vacuum to yield 8.68 g methoxy-PEG 5000 (zero
diol).
EXAMPLE 7
Synthesis of zero diol methoxypolyethylene glycol succinate from zero diol
methoxypolyethylene glycol
M-PEG-OH 5000 zero diol (4.7 g, 0.94 mmol) was dissolved in 100 ml toluene.
The solution was brought to reflux and a Dean-Stark trap was used to
remove any water. After I hour at reflux, a total of 80 ml toluene was
removed by distillation, and the vessel containing 20 ml toluene, was
allowed to cool under argon positive pressure. Succinic anhydride was
added (110 mg, 1.1 mmol), followed by 4-dimethylaminopyridine (137 mg,
1.12 mmol). Since the succinic anhydride did not dissolve, 10 ml of
anhydrous ethanol free chloroform was added, and the solution was held at
a reflux using an oven dried condenser. After 15 h at reflux, the solution
was cooled and then stirred with 10 g of cation exchange resin, filtered,
and the filtrate concentrated to obtain the title compound.
EXAMPLE 8
Synthesis of zero diol methoxypolyethylene glycol succinimidyl succinate
from zero diol methoxypolyethylene glycol succinate
A solution of M-PEG-succinate from Example 7 (4.15 g, 0.83 mmol) in 100 ml
of toluene was dried azeotropically. A portion of the toluene was
distilled off (60 ml, leaving 40 ml in the reaction flask) and
N-hydroxysuccinimide (100 mg, 0.87 mmol) was added, followed by the
careful addition of 30 ml of ethanol free anhydrous chloroform. An
additional 25 ml of the mixed solvent was removed by distillation and the
solution was allowed to cool at room temperature under argon. DCC was
added (200 mg, 9.7 mmol) and the solution was stirred. After 10 minutes,
DCU began to crystallize out. After stirring for two days, an additional
25 mg (0.22 mmol) of N-hydroxy succinimide was added. The dicyclohexyl
urea (DCU) slurry was filtered and the precipitate was washed with
toluene. The filtrate was concentrated by rotary evaporation giving an
additional precipitation of dicyclohexyl urea (DCU). The filtered
concentrate was added dropwise into one liter of anhydrous ether. The
precipitate was collected on a Whatman 9 cm 6F/F glass fiber filter and
then dried under high vacuum for 15 hours, to give 3.37 g Of M-PEG-SS.
Active ester content: 1.71.times.10.sup.-4 mol/g; HPLC indicated: 1.3%
M-PEG-S; other impurities: 1.2%; DCU none detected; total impurity: 3%.
EXAMPLE 9
Synthesis of zero diol PEG-SOD
Superoxide dismutase (1.33 ml of 75 mg/ml stock) was added to 18.67 ml of
reaction buffer (100 mM sodium phosphate, pH 7.8) and the solution was
brought to 30.degree. C. M-PEG-SS from Example 8 (300 mg) was added in one
portion and the pH was maintained at 7.8 by use of a pH stat. After 28
minutes the reaction pH became unchanging and the sample was concentrated
on Centrium centrifugal membrane of 10,000 MW cutoff. The concentrated
sample was exchanged in this manner with Dulbecco's PBS which had been
adjusted to pH 6.2 with 1M HCl. Five exchanges at a total of 60 ml were
performed. Size exclusion HPLC showed negligable high MW peak indicating
that the title compound contained negligable amounts of material derived
from diol (i.e., it was "zero diol").
The following examples illustrate the preparation of other biologically
active proteins covalently joined to PEG.
EXAMPLE 10
Synthesis of low diol methoxypolyethylene glycol-succinoyl-catalase
4.17 ml of an aqueous suspension of catalase containing 24.0 mg of protein
per ml was diluted with 15.84 ml of 0.1M sodium phosphate buffer, pH 7.8.
To this solution, magnetically stirred and heated to 30.degree. C., was
added 550 mg of low diol methoxy PEG-SS. The pH of the reaction mixture
was maintained at 7.8 using a Merrier DL25 titrator programmed in the pH
stat mode to add 0.5 normal sodium hydroxide solution as required. After
0.5 hour the reaction mixture was filtered through a 0.45 micron low
protein binding polysulfone filter and placed in two Amicon Centriprep 30
Concentrators (30K NMWL membrane) and buffer was exchanged several times
with Dulbecco's PBS. The retentate solution containing the low diol
PEG-catalase was then filtered through a 0.2 micron filter. Conjugate
formation was demonstrated by SEHPLC and gel electrophoresis.
EXAMPLE 11
Synthesis of low did PEG-Ovalbumin
503 mg of ovalbumin (Sigma) was dissolved in 50 g of 0.25M, pH 7.8
phosphate buffer at room temperature in a polyethylene beaker containing a
Telfon-coated magnetic stir bar. After stirring for 15 minutes, 1,900 g of
low diol M-PEG(5,000)-SS was added all at once. The pH of the reaction
mixture was controlled at 7.8 with a Mettler DL25 pH stat which added 0.5N
NaOH as needed. The reaction was allowed to continue for 1 h at room
temperature, and then the reaction mixture was diafiltered through an
Amicon YM30 membrane using a stirred cell device operated under 25 psi of
argon overnight in a refrigerator at 4.degree. C. After 800 ml of buffer
had beed diafiltered, the product was concentrated by ultrafiltration,
filtered through a 0.2 micron polysulfone filter, and vialed in sterile
glass vials to give 44.3 g of solution with a protein content of 10.5
mg/ml. The degree of protein modification was determined to be 71.4 % by
titration analysis of lysine amines.
EXAMPLE 12
Synthesis of low diol mPEG.sub.5K -S-Ovalbumin
10 ml of a cold 10 mg/ml solution of ovalbumin (Sigma, grade VI) in 0.25M
phosphate buffer, pH 7.4 was added to 382 mg of low diol mPEG.sub.5K -SS
and stirred at 5.degree. C for 16 hours. The product was purified in a
Centriprep 30 Concentrator (Amicon, 30K NMWL membrane) using Dulbelco's
PBS as the exchange buffer. The purified solution was filtered through a
0.2 .mu.m filter to give 6.539 g containing 13.8 mg/ml of 74% modified
(TNBS titration method) protein.
In a similar manner, 100 mg of ovalbumin was reacted with 283 mg of low
diol mPEG.sub.5K -SS giving 6.274 g containing 13.8 mg/ml of 74% modified
protein.
In a similar manner, 100 mg of ovalbumin was reacted with 190 mg of low
diol mPEG.sub.5K -SS giving 5.704 g containing 16.8 mg/ml of 67% modified
protein.
EXAMPLE 13
Synthesis of low diol mPEG.sub.5K -S-rhu-IL4
190 .mu.l of a 5.26 mg/ml solution of rhu-IL4 (Immunex) was diluted with
772 .mu.l of 0.1M borate buffer, pH 8.5. The rhu-IL4 solution was then
treated with 29.2 .mu.l of a 34 mg/ml solution of low diol methoxy PEG-SS
in DMF. After 1 hour and 20 minutes at room temperature the reaction
mixture was centrifuged and injected directly onto a preparative SEHPLC
column. The purified conjugate was shown to be essentially a single band
on gel electrophoresis.
EXAMPLE 14
Synthesis of low diol mPEG.sub.5K -S-NT
A solution containing 8.7 mg of neurotensin (NT) (BaChem) in 2.175 ml of
0.25 M phosphate buffer, pH 7.8 was added to 174 mg of low diol
mPEG.sub.5K -SS. The reaction mixture was kept at room temperature for
1.75 hours, then refrigerated. Pure mono-mPEG.sub.5K -S-NT was obtained
after separation from NT-PEG.sub.8K -NT by preparative reverse phase HPLC
on a C-8 column elating with a water/acetonitrile gradient.
Reactivity of Antibodies in Serum Treated with PEG-SOD
Enzyme-linked immunosorbent assays (ELISA) which detect circulating IgG
antibodies to PEG-SOD were used to assess the immunoreactivity of low did
PEG-SOD and high diol PEG-SOD.
Subjects enrolled in Phase I clinical studies received intravenous
injections of high diol PEG-SOD to produce antibodies in their serum.
Serum was then collected from patients. A statistical analysis of the
reactivity of circulating antibodies obtained from the Phase I study to
preparations of low and high did PEG-SOD both prepared with the same
percent modification of lysine amines was investigated. This analysis
examined the optical density differences among preparations across all
subjects treated with high diol PEG-SOD. The optical densities (OD) of the
post-treatment serum samples for low diol PEG-SOD were two to ten times
less than those for the high diol PEG-SOD. This unexpected result implies
that antibodies present in the post-treatment serum samples have little
reactivity to low diol PEG-SOD, and that the immunogenicity of low diol
PEG-SOD is considerably less than that of high diol PEG-SOD.
The method used in the analysis is as follows:
100 ml of 2.5 .mu.g/ml solution of high and low diol PEG-SOD was added to
wells of a CoBind microtiter plate. The plate was covered with Parafilm
and incubated overnight at room temperature in the dark. Unbound PEG-SOD
was removed from the wells of the microtiter plate by washing and
aspirating five times with distilled water followed by thorough blotting.
200 .mu.l of 1% gelatin in PBS was added to each well and incubated for
approximately 1 hour at 37.degree. C. The plates were then washed again
five times with PBS containing 0.05% Tween-20 and thoroughly blotted. 100
.mu.l of each diluted clinical sample was added in triplicate to the wells
of the microtiter plate and incubated for one hour at room temperature.
For each subject, a pre-dose sample and a post-dose sample was analyzed.
Plates were washed again as described above. 100 .mu.l of diluted goat
anti-human immunoglobulin conjugated to horseradish peroxidase was added
to each well and incubated for 1 hour at room temperature. Plates were
again washed as described above. 100 .mu.l of ABTS substrate solution was
added to each of the wells and incubated for approximately 20 minutes at
room temperature. Optical density readings for the individual wells were
taken using a dual wavelength setting (405 nm read, 490 nm reference) on a
BioTek EL 312 microplate reader. The magnitude of color development was
directly related to the amount of antibody present in the sample. The
results of the study are shown in Table 3.
TABLE 3
______________________________________
Relative Reactivity of ELISA-Positive Human Serum Samples with
Low Diol PEG-SOD and High Diol PEG-SOD Preparations
Mean OD Values.sup.a
Subject High Low
ID Sample Diol Diol
______________________________________
101 1.sup.b 0.039 0.045
2.sup.c 0.135 0.139
102 1.sup.b 0.061 0.072
2.sup.c 0.605 0.119
103 1.sup.b 0.043 0.038
2.sup.c 0.707 0.393
104 1.sup.b 0.092 0.119
2.sup.c 0.411 0.254
105 1.sup.b 0.037 0.051
2.sup.c 0.331 0.114
106 1.sup.b 0.316 0.235
2.sup.c 0.595 0.242
107 1.sup.b 0.360 0.132
2.sup.c 0.896 0.771
108 1.sup.b 0.123 0.103
2.sup.c 0.354 0.281
109 1.sup.b 0.082 0.073
2.sup.c 0.214 0.113
110 1.sup.b 0.296 0.075
2.sup.c 0.728 0.089
111 1.sup.b 0.066 0.055
2.sup.c 0.355 0.119
112 1.sup.b 0.064 0.208
2.sup.c 0.570 0.388
113 1.sup.b 0.134 0.073
2.sup.c 0.384 0.086
114 1.sup.b 0.049 0.048
2.sup.c 0.302 0.057
115 1.sup.b 0.077 0.081
2.sup.c 0.678 0.307
116 1.sup.b 0.079 0.071
2.sup.c 0.675 0.213
117 1.sup.b 0.077 0.087
2.sup.c 0.335 0.161
118 1.sup.b 0.209 0.121
2.sup.c 0.531 0.152
119 1.sup.b 0.160 0.141
2.sup.c 0.423 0.145
120 1.sup.b 0.155 0.097
2.sup.c 0.478 0.189
______________________________________
.sup.a = 3
.sup.b = Day 1 Predose Sample
.sup.c = Post Dose Sample
Utility of the PEG-SOD formulations resides in the prevention and treatment
of oxidative injury in a clinical setting.
Free oxygen radicals have been postulated as important mediaters in a broad
spectrum of clinical disorders, including life-threatening disease
processes such as carcinogenesis and aging, and in ischemia-reperfusion
injury. Studies have shown that free oxygen radicals (O.sub.2.sup.-.)
cause chemical modification of proteins, lipids, carbohydrates and
nucleotides. Fluxes of O.sub.2.sup.-. have been shown to kill bacteria,
inactivate viruses, lyse erthrocytes, destroy granulocytes, damage
myoblasts in culture, depolymerize hyaluronate, modify low-density
lipoprotein and damage DNA. Hydrogen peroxide may potentiate the effects
of proteases (e.g., trypsin), resulting in a modification of protein
substrates such as fibrinogen, hemoglobin and glomerular basement
membrane, so that they become very susceptible to proteolysis. Other
cytotoxic consequences of free radical formation and accumulation include
alterations in membrane integrity and permeability, loss of enzyme
activity due to denaturation of proteins and increased inflammatory
response.
Endogenous scavenger enzymes such as superoxide dismutase (SOD) and
catalase (CAT) normally remove toxic oxygen intermediates before they can
cause tissue injury. However, during reperfusion, and in certain other
clinical settings, the endogenous enzyme defense mechanisms may be
overwhelmed. Supplementing these enzymes is of therapeutic benefit. Free
radical scavengers such as SOD have been reported to attenuate
ischemic-related damage accompanying reperfusion in several animal models.
See, for example: Shlafer, M., Kane, P. F., Kirsch, M. M., "Superoxide
Dismutase Plus Catalase Enhances the Efficacy of Hypothermic Cardioplegia
to Protect the Globally Ischemic, Reperfused Heart", J. Thorac Cardiovasc.
Surg. 1982;83:630; Shlafer, M., Kane, P. F., Wiggins, V. Y., Kirsch, M.
M., "Possible Role of Cytotoxic Oxygen Metabolites in the Pathogenesis of
Cardiac Ischemic Injury", Circ. 1982;66(Suppl I):1-85; Casale, A. S.,
Bulkley, G. B., Bulkley, B. H., Flaherty, J. T., Gott, V. L., Gardner, T.
J., "Oxygen Free-Radical Scavengers Protect the Arrested, Globally
Ischemic Heart Upon Reperfusion", Surg. Forum. 1983;34:313; Stewart, J.
R., Blackwell, W. H. A., Crute, S. L., Loughlin, V., Hess, M. L.,
Greenfield, L. J., "Prevention of Myocardial Ischemia/Reperfusion Injury
with Oxygen Free Radical Scavengers", Surg. Forum. 1982;33:317;
Pryzyklenk, K., Kloner, R. A., "Superoxide Dismutase Plus Catalase Improve
Contractile Function in the Canine Model of the Stunned Myocardium", Circ.
Res. 1986;58:148-156; and Beckman, J. S., Minor, R. L. Jr., White, C. W.,
Repine, J. E., Rosen, G. M., Freeman, B. A., "Superoxide Dismutase and
Catalase Conjugated to Polyethylene Glycol Increases Endothelial Enzyme
Activity and Oxidant Resistance", J. Biol. Chem. 1988;263(14):6884-6892.
In a method aspect, the present invention provides for preventing and/or
treating oxidative injury in a mammal which treatment comprises
administering to said mammal a therapeutically effective amount of a
formulation of PEG-SOD in a pharmaceutically acceptable carrier. The
Amount of units of SOD to be administered parenterally will depend on the
particular condition treated, the age, weight and other characteristics of
the patient, which are to be judged by the physician. A dosage range of
from about 2,000 to about 50,000 units per kg of body weight is envisaged
for administration, while a range of from about 5,000 to about 25,000
units per kg of body weight is preferred.
While preferred embodiments of the invention have been described and
illustrated in the specification, it is to be understood that such is
merely illustrative of the underlying concept and features of the
invention and are not to be limiting of the scope of the invention and the
appended claims.
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